Brucellosis 2003 proceedings - PHIDIAS

Short Oral Communications
Vaccines
VO9- WANING trans COMPLEMENTATION OF ROUGHNESS IN A Brucella
melitensis wboA purE DUAL MUTANT: A POTENTIAL FOR LIVE VACCINES
AND HETEROLOGOUS ANTIGEN DELIVERY.
M. Nikolich, M. Izadjoo, C. Fernandez-Prada, E. Penn and D. Hoover. Walter Reed Army Institute of
Research, Silver Spring, MD, USA.
Brucella melitensis strain WRRP1 was constructed by deleting wboA and
purEK loci in the B. melitensis strain 16M genome by allelic exchange. This rough
purine auxotroph survived in significantly lower numbers in human monocyte-derived
macrophages (MDMs) compared with either the ∆wboA parent strain WRR51 or with
∆purEK strain WR201. WRRP1 was also severely attenuated for virulence in BALB/c
mice after intraperitoneal or oral introduction, where it persisted for less than one
week. Provision of intact wboA on a pBBR1-derived plasmid in WRR51 restored
both smoothness and a level of replication in MDMs indistinguishable from that of
strain 16M. As expected, like trans complementation of wboA in WRRP1 restored
smoothness and led to increased survival in MDMs in numbers not statistically
different from those of ∆purEK strain WR201. However, a small subset of colonies
recovered from MDMs infected with wboA-complemented WRRP1 lost the
complementing plasmid and reverted to the rough phenotype. Plasmid loss was
replicated in broth culture and preceded to completion in passage over two months.
Loss of the complementing plasmid was also observed in colonies recovered from
BALB/c mice orally infected with complemented WRRP1. Complemented WRRP1
persisted in the spleens of these mice for six weeks, compared with clearance by one
week of the uncomplemented dual mutant. The duration of spleen persistence was
not unlike that of ∆purEK strain WR201, which in most experiments was cleared
before eight weeks. However, complemented WRRP1 persisted in spleens in
significantly lower numbers at all timepoints than was seen with WR201. This
reduction in bacterial load indicated loss of the complementing plasmid in a
subpopulation of the bacteria within the host, resulting in uncomplemented WRRP1
attenuation and rapid clearance. The extended persistence of wboA-complemented
WRRP1 in lower numbers in the host offers the potential for a safer live vaccine
alternative to strain WR201 that may retain the protective qualities of the ∆purEK
genotype. Loss of the complementing plasmid and reversion to roughness also
provides a period of exposure of rough surface antigens within the host that may
enhance immunogenicity. Additionally, wboA-complementation of WRRP1 clearly
provided selective pressure for retention of the complementing plasmid; this selection
was most pronounced in mice and MDMs, but was even observed in broth culture.
The strong in vivo selection for the complementing plasmid can be exploited to
maintain the expression within mammalian hosts of antigens from heterologous
pathogens co-delivered on the plasmid in WRRP1, or a derivative live attenuated
Brucella carrier. We demonstrated this potential of our system for foreign antigen
delivery in vaccinees by expressing a heterologous gene encoding Green
Fluorescent Protein from the wboA-complementing plasmid in BALB/c mice over a
six-week period.
Brucellosis 2003 International Research Conference
81